Studies designed to elucidate the mechanisms of action of inducing agents include 1) Identification of chemical compounds more active as inducers, 2) Identification of the molecular portion of the inducing molecule which confers activity, and 3) Determination of the cellular uptake of inducer, its intracellular location, and its metabolism. Of the many chemical compounds demonstrated to induce differentiation, a common characteristic is the possession of both a hydrophilic, polar group combined with a hydrophobic portion in the molecule. The most potent inducer we have identified is the hexamethylene bisacetamide (HMBA) which induces all cells to differentiate at 5mM. Early biochemical and biological events resulting from culture with inducers include alterations in cell cycle traverse, changes in chromatin and DNA structure, "commitment" to differentiation, and initiation of alpha and beta globin gene transcription. Transient prolongation of G1 and increased alkaline lability of the DNA are detected within the first 24 hours of culture, as is "commitment" to differentiate assayed at the single cell level, and initiation of globin mRNA transcription. While numerous chemical compounds induce differentiation, differential expression of the beta globin genes (beta maj and beta min) varies with the inducer employed.